During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, diseases and herbicides, and to modify biosynthetic processes to change the quality of plant products (Knutson et al., PNAS, USA 89, 2624-2628, (1992); Piorier et al., Science, 256, 520-523, (1992); Vasil et al., Bio/Technology, 10, 667-674, (1992)). However, the availability of an efficient transformation method to introduce foreign DNA has been a substantial barrier for most monocot species, including maize, rice, oat, barley, and particularly wheat.
Two alternative transformation methods are currently used for monocot species: direct DNA transfer into isolated protoplasts and microprojectile-mediated DNA delivery (Shimamoto et al., Nature, 338, 274-276, (1989); Fromm et al., Bio/Technology, 8, 833-839, (1990)).
The protoplast methods have been widely used in rice, where DNA is delivered to the protoplasts through liposomes, PEG, and electroporation. While a large number of transgenic plants have been recovered in several laboratories (Shimamoto et al., (1989); Datta et al., Bio/Technology, 8, 736-740, (1990)), the protoplast methods require the establishment of long-term embryogenic suspension cultures. Some regenerants from protoplasts are infertile and phenotypically abnormal due to the long-term suspension culture (Davey et al., J. of Exp. Botany, 42, 1129-1169, (1991); Rhodes et al., Science, 240, 204-207, (1988)).
The microprojectile-mediated DNA delivery method may use immature embryos or immature embryo derived calli as target tissues. Transgenic plants have been recovered from the microprojectile bombardment method in maize, oat, barley and wheat (Gordon-Kamm et al., Plant Cell, 2, 603-618, (1990); Somers et al., Bio/Technology, 10, 1589-1594, (1992); Wan et al., Plant Physiol., 104, 37-48 (1994); Vasil et al. (1992)).
The microprojectile bombardment method generally takes 10 to 15 months to obtain transgenic plants (Gordon-Kamm et al., (1990); Vasil et al. (1992)). Even with the more recent improvements in transformation methods using immature embryos as target tissues, it still requires 4 to 6 months to recover transgenic plants (Weeks et al., Plant Physiol., 102, 1077-1084, (1993); Vasil et al., (1992); Vasil et. al., Bio/Technology, 11, 1153-1158 (1993); Becker et al., Plant J., 5, 299-307, (1994). Moreover, these methods suffer frequently from a loss in fertility in the recovered plants (Vasil et al., (1993); Becker et al., (1994)). Furthermore, the transformation frequency by these methods is very low, about one event from every thousand bombarded embryos. This transformation efficiency is too low for genetic studies and for commercial applications.
Thus, there is a need not only for a more rapid method of regenerating transformed plant tissue, there is also a need for a method that retains fertility in the resulting plants and produces a higher transformation efficiency.